The vitamin A metabolite, all-trans-retinoic acid (ATRA), plays essential roles in a multitude of biological processes throughout life and has shown promising results in several therapeutic settings. However, exposure to either reduced or excess levels of ATRA during pregnancy can result in abnormalities in embryonic development. The specific goal of this project is to determine the factors that control the formation of ATRA in vivo. The long-term goal is to develop therapies based on the manipulation of the endogenous levels and activity of ATRA in diseased tissues. The central hypothesis is that ATRA regulates its own synthesis via a homeostatic feedback mechanism by controlling the balance of the oxidation of retinol versus the reduction of retinaldehyde. Current studies have largely ignored the role of enzymes that carry out the reduction of retinaldehyde in the regulation of ATRA levels. The overall objective of this application is to determine the role and regulation of DHRS3, shown here to be a pivotal enzyme in vitamin A metabolism. This hypothesis is based on preliminary results derived from the characterization of a Dhrs3-deficient mouse model. These results indicate that 1) DHRS3 is the predominant enzyme responsible for the reduction of retinaldehyde during development and 2) the reduction of retinaldehyde by DHRS3 is indispensable for ATRA homeostasis and normal embryonic development. Testing of the central hypothesis will be pursued in three specific aims: Aim 1 studies will establish 1) the enzymatic properties of DHRS3, 2) the mechanism of regulation of Dhrs3 by ATRA, and 3) the spatial- temporal pattern of the expression of Dhrs3 during development. This knowledge is essential for understanding the physiological role of DHRS3. Studies described in Aim 2 will determine the role of DHRS3 in vitamin A metabolism based on the working hypothesis that DHRS3 is the main enzyme responsible for reducing retinaldehyde during embryogenesis. Aim 2 will be pursued by establishing the effect of Dhrs3-ablation on the metabolism of retinol and provitamin A carotenoids by 1) LC-UV and LC-MS/MS analysis of the levels of retinoids, and 2) assaying the expression of ATRA-target genes in the tissues of Dhrs3-/- embryos. Aim 3 will test the hypothesis that the developmental defects caused by ablation of Dhrs3 are a result of altered ATRA metabolism. Preliminary studies indicate that Dhrs3-/- embryos have defects in anterior-posterior patterning, cardiac and skeletal development, and are non-viable. Aim 3 will be pursued by characterizing the pathways responsible for the observed developmental defects in relation to altered ATRA formation. Therefore, the effect of diet, genetic background and inhibition of vitamin A metabolism on the manifestation of developmental defects associated with Dhrs3-ablation will be examined. These studies aim to further establish the processes responsible for the regulation of ATRA formation in vivo and contribute to a framework for the long-term goal of developing approaches to prevent and treat congenital and inherited disorders associated with altered ATRA-signaling.